#9-Tetrahydrocannabinol-Induced Apoptosis in JurkatLeukemia T Cells Is Regulated by Translocationof Bad to Mitochondria

Wentao Jia,2 Venkatesh L. Hegde,1 Narendra P. Singh,1 Daniel Sisco,1

Steven Grant,3 Mitzi Nagarkatti,1 and Prakash S. Nagarkatti1

1Department of Pathology, Microbiology, and Immunology, University of South CarolinaSchool of Medicine, Columbia, South Carolina and Departments of 2Pharmacologyand Toxicology and 3Medicine, Medical College of Virginia Campus,Virginia Commonwealth University, Richmond, Virginia

AbstractPlant-derived cannabinoids, including

#9-tetrahydrocannabinol (THC), induce apoptosis in

leukemic cells, although the precise mechanism remains

unclear. In the current study, we investigated the effect

of THC on the upstream and downstream events that

modulate the extracellular signal-regulated kinase (ERK)

module of mitogen-activated protein kinase pathways

primarily in human Jurkat leukemia T cells. The

data showed that THC down-regulated Raf-1/mitogen-

activated protein kinase/ERK kinase (MEK)/ERK/RSK

pathway leading to translocation of Bad to mitochondria.

THC also decreased the phosphorylation of Akt.

However, no significant association of Bad translocation

with phosphatidylinositol 3-kinase/Akt and protein

kinase A signaling pathways was noted when treated

cells were examined in relation to phosphorylation

status of Bad by Western blot and localization of Bad to

mitochondria by confocal analysis. Furthermore, THC

treatment decreased the Bad phosphorylation at Ser112

but failed to alter the level of phospho-Bad on site

Ser136 that has been reported to be associated with

phosphatidylinositol 3-kinase/Akt signal pathway. Jurkat

cells expressing a constitutively active MEK construct

were found to be resistant to THC-mediated apoptosis

and failed to exhibit decreased phospho-Bad on Ser112

as well as Bad translocation to mitochondria. Finally,

use of Bad small interfering RNA reduced the expression

of Bad in Jurkat cells leading to increased resistance

to THC-mediated apoptosis. Together, these data

suggested that Raf-1/MEK/ERK/RSK-mediated Bad

translocation played a critical role in THC-induced apoptosis

in Jurkat cells. (Mol Cancer Res 2006;4(8):549–62)

IntroductionCannabinoids, the biologically active constituents of mari-

juana (Cannabis sativa), produce a wide spectrum of central

and peripheral effects, such as alterations in cognition and

memory, analgesia, anticonvulsion, anti-inflammation, and

alleviation of both intraocular pressure and pain relief (1).

There has been a growing interest in cannabinoids since the

cloning of two subtypes of the cannabinoid receptors, CB1 (2)

and CB2 (3). The CB1 receptor is mainly expressed in the

central nervous system, whereas the CB2 receptor is predom-

inantly expressed in immune cells (4). Both cannabinoid

receptors are coupled to heterotrimeric Gi/o proteins and interact

with the mitogen-activated protein kinases (MAPK), particu-

larly the extracellular signal-regulated kinase (ERK; ref. 5).

At almost the same time, endogenous ligands for these

receptors, capable of mimicking, to some extent, the phar-

macologic actions of marijuana’s psychoactive principle D9-tetrahydrocannabinol (THC), have been discovered (6).

Numerous studies have shown that THC can modulate the

functions of immune cells (7). More recently, we reported that

the immunosuppressive property of THC can be attributed, at

least in part, to its ability to induce apoptosis in T cells and

dendritic cells through ligation of CB2 receptors and that the

latter was regulated by activation of nuclear factor-nB (8),recruiting both intrinsic and extrinsic pathways of apoptosis.

Interestingly, we also found that THC and other cannabinoids

could induce apoptosis in transformed murine and human

T cells (9), including primary acute lymphoblastic human

leukemia cells, and furthermore that the treatment of mice

bearing a T-cell leukemia with THC could cure f25% of themice (10). These findings are consistent with studies showing

that THC and other cannabinoids can induce apoptosis in a

variety of tumor cell lines, thereby raising the possibility of the

use of cannabinoids as novel anticancer agents (11).

The precise mechanism through which cannabinoids induce

apoptosis is under active investigation and may vary based on

cell type. In normal and transfected neural cells, vascular

endothelial cells, and Chinese hamster ovary cells, cannabinoid

treatment was shown to induce activation of ERK (12), c-Jun

NH2-terminal kinase (JNK), and p38 (13, 14). In contrast, it

was shown that cannabinoids were cytotoxic in leukemic cells

and that they inhibited neuronal progenitor cell differentiation

through attenuation of the ERK pathway (15). In glioma cells,

THC was shown to induce apoptosis via ceramide generation

Received 10/3/05; revised 6/19/06; accepted 6/21/06.Grant support: NIH grants R01DA016545, R01ES09098, R01AI053703,R01AI058300, R01HL058641, R21DA014885, and P30CA16059.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Prakash S. Nagarkatti, Department of Pathology,Microbiology, and Immunology, University of South Carolina School ofMedicine, Columbia, SC 29208. Phone: 803-733-3180; Fax: 803-733-3335.E-mail: pnagark@gw.med.sc.eduCopyright D 2006 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-05-0193

Mol Cancer Res 2006;4(8). August 2006 549

(16). However, cannabinoids can also block ceramide-induced

apoptosis of normal astrocytes (17). Together, such studies

suggest that the precise signaling pathways that are evoked by

cannabinoid receptor activation in normal and transformed cells

may vary based on cell type and that such activation could lead

to the generation of survival or death signals.

In the current study, we investigated the molecular

mechanisms underlying apoptosis induced by THC, specifi-

cally addressing the role of MAPK signaling. Treatment with

THC caused interruption of the MAPK/ERK kinase (MEK)/

ERK signaling module that was required for apoptotic

lethality, and this event possibly played an important

functional role in mediating THC-induced translocation of

Bad to mitochondria.

ResultsTreatment of Jurkat Cells with THC Induces Suppressionof the Raf-1/MEK/ERK Cytoprotective Signaling Pathwaythrough the Signaling of the Cannabinoid Receptors

To gain insights into the functional role of cannabinoid

receptor pathway in THC-mediated lethality, Jurkat cells were

pretreated with either SR141716 (CB1 antagonist) or

SR144528 (CB2 antagonist) in the absence or presence of

THC for a designated period (6-30 hours), after which the

percentage of cells displaying the morphologic features of

apoptosis was determined by the Wright-Giemsa-stained

cytospin preparation. THC caused dose-dependent apoptosis

in Jurkat cells (data not shown) with a very substantial increase

in cell death detected at 10 Amol/L (Fig. 1A). Time courseanalysis revealed that exposure to SR141716 (CB1 antagonist)

or SR144528 (CB2 antagonist) individually at the concentration

of 1 or 2 Amol/L were minimally toxic over 30-hour treatmentinterval, whereas treatment with 10 Amol/L THC alone causedf50% apoptosis (Fig. 1A). However, when cells werepretreated with either SR141716 or SR144528 followed by

exposure to THC, there was a significant reduction in apoptosis

by 6 hours and a very substantial reversal in lethality after 12

hours (Fig. 1A). Very similar results were obtained when THC-

treated cells were evaluated for combined early and late

apoptotic cells by Annexin V/propidium iodide (PI) analysis

(Fig. 1B). In this assay, cells stained for Annexin V alone are

considered to be early apoptotic cells and those stained for

Annexin V and PI represent late apoptotic cells. We also

analyzed the cells for loss of mitochondrial membrane potential

(MMP; Dcm) as described (10) and designated them as cellswith ‘‘low’’ 3,3-dihexyloxacarbocyanine iodide uptake

(Fig. 1B). Next, we measured the levels of CB1 and CB2

receptors in Jurkat cells and compared them with other tumor

cell lines, such as the T-cell lymphoma Hut78 and the glioma

U251 known to express CB1 receptors. As shown in Fig. 1C,

Jurkat and Hut78 but not U251 cells expressed significant

levels of CB2 mRNA. In addition, when CB1 mRNA levels

were analyzed, Jurkat cells were found to express low levels.

Interestingly, when all cells were cultured with THC, CB1 and

CB2 receptor expression was significantly increased. These

data explained why in Jurkat cells not only CB2 antagonist but

also CB1 antagonist were able to partially block THC-induced

apoptosis.

To further characterize the downstream signaling pathways

that trigger apoptosis following activation of cannabinoid

receptors, Jurkat cells were pretreated with either CB1 or

CB2 antagonists in the absence or presence of THC for 12

hours. As shown in Fig. 1D, THC treatment of Jurkat cells

caused reduced phosphorylation of Raf-1, whereas total protein

levels remained constant. In addition, exposure of cells to THC

diminished the levels of phosphorylation of MEK1/2, with the

total MEK1/2 expression remaining unchanged. Similarly,

phosphorylation of ERK1/2 was largely decreased in THC-

treated cells, but total ERK1/2 expression remained unper-

turbed. In contrast, when cells were pretreated with CB1 or

CB2 antagonists, the effects of THC on each of these proteins

were largely reduced. None of the THC concentrations tested

induced alterations in phospho-p38 or phospho-JNK. In

addition, no changes were observed in levels of total p38 or

JNK. Consequently, we evaluated the levels of phospho-

p90RSK (Thr359/Ser363), phospho-p90RSK (Ser380), phospho-

p90RSK (Thr573), and total p90RSK following exposure of

Jurkat cells to THC in the absence or presence of CB1/2

antagonists (Fig. 1D). THC caused a reduction in p90RSK

phosphorylation involving both Thr359- and Ser363-specific

phosphorylation sites. However, when cells were pretreated

with CB1 or CB2 antagonist, the effects of THC on both

phosphorylation sites of this protein were significantly

reversed. No changes were detected in levels of total p90RSK

or phospho-p90RSK on sites Ser380 and Thr573. Attempts also

were made to extend ERK inactivation to other human tumor

cell lines. As shown in Fig. 1E, an 18-hour exposure of Molt4,

Hut78, and SupT1 cells to THC resulted in a marked reduction

in the levels of phospho-ERK1/2, with the total ERK1/2

expression remaining unchanged. Finally, essentially similar

results were obtained when the effects of THC were examined

in kinase activity. A decrease in ERK activity was detectable in

Jurkat cells after treatment with THC beginning at hour 1, and

this effect was even more pronounced at 12 hours (Fig. 1F).

Taken together, these data suggest that suppression of the Raf-

1/MEK/ERK cytoprotective signaling pathway by THC may

play an important functional role in the induction of apoptosis

in Jurkat cells as well as in other tumor cell lines tested.

Caspase-Independent Events in Raf-1/MEK/ERKSignaling

To determine the role caspase activation in THC-mediated

perturbations in ERK signaling, Jurkat cells were pretreated

with pan-caspase inhibitor Z-VAD-FMK in the presence or

absence of THC. As shown in Fig. 2A, caspase inhibition failed

to alter the THC-mediated down-regulation of phosphorylation

of Raf-1, MEK1/2, ERK1/2, and RSK (Thr359/Ser363). No

changes were noted in the levels of total Raf-1, MEK1/2,

ERK1/2, and RSK or phospho-p90RSK on sites Ser380 and

Thr573. These results indicated that in Jurkat cells down-

regulation of the Raf-1/MEK/ERK/RSK axis represents an

early consequence of the treatment of cells with THC and that it

proceeds in a caspase-independent manner. As shown in

Fig. 2B, exposure of Jurkat cells to Z-VAD-FMK significantly

inhibited THC-induced procaspase-10, procaspase-2, procas-

pase-8, procaspase-9, procaspase-3, and Bid processing as well

as poly(ADP-ribose) polymerase (PARP) degradation. It should

Jia et al.

Mol Cancer Res 2006;4(8). August 2006

550

be noted that treatment of Jurkat cells with Z-VAD-FMK alone

did not alter the expression of any of the molecules under

investigation (data not shown).

Antiapoptotic Effect of Phorbol 12-Myristate 13-AcetateDepends on ERK Activation

To confirm the possible role of ERK activation in THC-

induced apoptosis, Jurkat cells were pretreated with 10 nmol/L

phorbol 12-myristate 13-acetate (PMA), a potent activator of

ERK (18, 19). Next, the cells were treated with10 Amol/L THCfor a total of 12 hours or left untreated. Parallel studies were

done on cells precultured with 25 Amol/L U0126, a MEKinhibitor. The extent of apoptosis was then determined by

terminal deoxynucleotidyl transferase–mediated dUTP end

labeling (TUNEL) assay. Treatment of cells with THC alone

for 12 hours caused f43% apoptosis, which was partially

FIGURE 1. Effects of THC on Raf-1/MEK/ERK signaling in Jurkat cells. A. Jurkat cells were pretreated with either 1 Amol/L SR141716 or 2 Amol/LSR144528 in medium containing 10% FBS in the absence or presence of 10 Amol/L THC for a designated period (6-30 hours), after which the percentageof apoptosis was determined by examining Wright-Giemsa-stained cytospin preparations as described in Materials and Methods. Points, mean percentapoptosis of at least three separate experiments done in triplicate; bars, SD. B. Cells were treated as described in A for 12 hours, after which the extentof apoptosis and loss of MMP were monitored by Annexin V/PI staining and 3,3-dihexyloxacarbocyanine iodide uptake, respectively. Columns, mean percentapoptosis of three separate experiments done in triplicate; bars, SD. C. Expression of CB1 and CB2 was determined by reverse transcription-PCR analysis.Total RNA was isolated from Jurkat, Hut78, and U251 tumor cells, which were either left untreated or treated with THC. mRNA was reverse transcribed andamplified by PCR with primers specific for CB1 and CB2. Photograph of ethidium bromide–stained amplicons. D. Cells were treated as described in A for12 hours, after which the Western analysis was used to monitor expression of phospho-Raf-1, Raf-1, phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2,phospho-RSK (Thr359/Ser363), phospho-RSK (Ser380), phospho-RSK (Thr573), RSK, phospho-p38, p38, phospho-JNK, and JNK. h-Actin served as loadingcontrol. E. Molt4, Hut78, and SupT1 cells were either left untreated or treated with THC at designated concentrations (7.5, 5, and 10 Amol/L, respectively) inmedium containing 10% FBS for 18 hours, after which the levels of phosphor-ERK1/2 and total ERK1/2 were monitored by Western blot. ERK1/2 served asloading control. F. Jurkat cells were either left untreated or treated with 10 Amol/L THC for a designated interval (from 0.5 to 12 hours), after which an ERKkinase assay was done by immunoprecipitation of ERK using a monoclonal phospho-ERK1/2 (Thr202/Tyr204). The immunoprecipitate was subsequentlyincubated with an Elk-1 fusion protein in the presence of ATP, which allowed precipitated phospho-ERK to phosphorylate Elk-1, a major substrate ofphospho-ERK. D, DMSO. Elk-1 served as loading controls. Representative of a minimum of three separate experiments.

Mechanism of THC-Induced Apoptosis

Mol Cancer Res 2006;4(8). August 2006

551

blocked in the presence of either CB1 or CB2 antagonist,

thereby confirming the involvement of CB1 and CB2 receptors

in apoptosis (Fig. 3A). In addition, THC-induced apoptosis was

also partially blocked by PMA (Fig. 3A). Moreover, treatment

of cells with U0126 alone caused significant levels of apoptosis

consistent with our hypothesis that MEK inhibition in Jurkat

cells can lead to cell death. Furthermore, the combination of

THC plus U0126 caused a marked increase in apoptosis, which

was more than that seen when either of these compounds was

used alone. As expected, treatment with PMA plus U0126

caused a significant inhibition in apoptosis when compared

with the use of U0126 alone. To characterize interaction

between THC and U0126 more rigorously and over a range of

drug concentrations at a fixed ratio (1:2.5), median dose effect

analysis was used. When the extent of apoptosis was

determined, combination index values considerably less than

1.0 were obtained (Fig. 3B), corresponding to highly

synergistic interaction. These data together suggest that THC-

induced ERK inactivation may play a critical role in apoptosis

in Jurkat cells.

Next, we determined protein levels of phospho-ERK to see

what protective action was mediated by PMA in THC-treated

cells. As shown in Fig. 3C, incubation with PMA alone induced

increased phosphorylation of ERK1/2. When cells were exposed

to PMA followed by THC, there was significant reversal in

phospho-ERK1/2 levels. In contrast, treatment with U0126

alone caused a significant decrease in phospho-ERK1/2,

whereas the combination of U0126 plus THC resulted in

essentially the complete disappearance of phospho-ERK1/2.

PMAwas a potent activator of ERK as has also been reported by

others (18, 19), inasmuch as it reversed the effect of the

combined treatment of U0126 plus THC on ERK1/2. These data

were corroborated determining the levels of the phosphorylation

of ERK1/2 by densitometric analysis (Fig. 3D). Finally, the

effect of these agents, alone and in combination, were examined

in relation to caspase cascades in THC-treated cells (Fig. 3E).

Whereas U0126 alone had a demonstrable effect on the

activation of all caspases tested as well as on the cleavage of

caspases and PARP, treatment with THC alone caused much

more cleavage of each of these proteins, consistent with the

increased levels of apoptosis observed previously. Treatment

with PMA blocked the THC-induced activation of caspases and

other markers of apoptosis. Caspase analysis using U0126 alone,

PMA plus U0126, and U0126 plus THC was all consistent with

the above observation on the ability of U0126 to enhance and

PMA to block apoptosis in THC-treated Jurkat cells.

Enforced Activation of MEK1/ERK Substantially BlocksTHC-Mediated Caspase Activation, DNA Fragmentation,and Apoptosis

To further define the functional role of MEK/ERK, a Jurkat

cell line that inducibly expresses a constitutively active MEK1

construct (Mek/30) under the control of a doxycycline-

responsive promoter was employed. As shown in Fig. 4A,

exposure to THC in the absence of doxycycline resulted in

apoptosis in f50% of cells, whereas apoptosis was markedlyreduced in the presence of doxycycline at 12 hours (P < 0.05),

and these effects were even more pronounced at 24 hours

(P < 0.005; data not shown). Similar results were obtained with

a second MEK1-inducible clone (Jurkat Mek/6; data not

shown). Western analysis revealed that cells cultured in the

absence of doxycycline displayed minimal expression of a

hemagglutinin tag and modest basal expression of phospho-

MEK (Fig. 4B). However, when cells were cultured in the

presence of doxycycline, a pronounced increase in expression

of the hemagglutinin tag was noted along with substantial

increases in expression of phospho-MEK, phospho-ERK, and

phospho-RSK (Thr359/Ser363). Enforced activation of MEK

also diminished THC-mediated activation of procaspase-8,

procaspase-9, procaspase-3, and Bid processing as well as

PARP degradation. Analogous to results obtained with the

inducible MEK system, in the absence of doxycycline, THC

treatment alone resulted in the induction of DNA fragmentation

(Fig. 4C). When cells were cultured in the presence of doxy-

cycline, DNA degradation was significantly blocked. Together,

these data suggest that Raf-1/MEK/ERK/RSK pathways play

an important functional role in THC-induced apoptosis.

Pertussis Toxin Pretreatment Prevents THC-Induced CellDeath

Next, we examined the relationship between receptor-

mediated activation of the G protein and THC-mediated

apoptosis. To this end, Jurkat cells were exposed to either

50 or 100 ng/mL pertussis toxin (PTX) for 16 hours in the

absence or presence of 10 Amol/L THC for an additional 12hours. As shown in Fig. 5A, pretreatment with PTX caused a

significant inhibition in THC-induced apoptosis. In related

studies, the effect of PTX was also monitored with respect to

MAPK signaling and the activation of apoptotic regulatory

proteins induced by THC. Exposure of cells to PTX reversed

the THC-mediated reduction in expression of phospho-Raf-1

as well as phosphorylation of MEK1/2, ERK1/2, and RSK

FIGURE 2. Effect of exposure of Jurkat cells to pan-caspase inhibitor,Z-VAD-FMK. Jurkat cells were pretreated with 20 Amol/L Z-VAD-FMK inmedium containing 10% FBS in the presence or absence of 10 Amol/LTHC for a total 12 hours, after which Western analysis was done tomonitor expression of (A) phospho-Raf-1, Raf-1, phospho-MEK1/2,MEK1/2, phospho-ERK1/2, ERK1/2, phospho-RSK (Thr359/Ser363), phos-pho-RSK (Ser380), phospho-RSK (Thr573), and RSK and after 24 hours tomonitor expression of (B) procaspase-10, procaspase-2, procaspase-8,Bid, procaspase-9, procaspase-3, and PARP or cleaved fragments (CF ).h-Actin served as a loading control. Representative experiment. Twoadditional studies yielded similar results.

Jia et al.

Mol Cancer Res 2006;4(8). August 2006

552

(Thr359/Ser363; Fig. 5B). Similarly, PTX treatment reversed other

THC-mediated effects, including cleavage of procaspase-10,

procaspase-2, procaspase-8, procaspase-9, procaspase-3, and

Bid and degradation of PARP (Fig. 5B). Together, these data

suggest a role for G-protein signaling in THC-induced MAPK

signaling, caspase activation, and apoptosis.

THC Down-Regulates the Raf-1/MEK/ERK/RSK SignalingPathway and Triggers Mitochondrial Localization of Bad

Recently, the MAPK-activated RSK was shown to promote

cell survival through phosphorylation and inactivation of the

proapoptotic Bcl-2 family member, Bad (18). In addition,

mitochondrial membrane-based protein kinase A (PKA) and

Akt, a kinase activated by growth factors through a phospha-

tidylinositol 3-kinase (PI3K)–dependent mechanism, could

also be implicated in Bad phosphorylation. To this end, we

examined whether Bad played a role in THC-induced apoptosis

involving Akt and PKA signaling pathways. First, Jurkat cells

were cultured with either 15 Amol/L LY294002, a PI3Kinhibitor, or 2 Amol/L H-89, a PKA inhibitor. Next, the cellswere treated with 10 Amol/L THC for a total of 12 hours or leftuntreated. The percentage of apoptotic cells was then

determined by examining Wright-Giemsa-stained cytospin

preparations. As shown in Fig. 6A, coadministration of

LY294002 at a concentrations of 15 Amol/L, which wasminimally toxic alone, resulted in apoptosis in majority of

THC-treated cells. A very similar pattern was noted when the

extent of apoptosis was determined by TUNEL assay (data not

FIGURE 3. Role of ERK action in the regulation of THC-induced apoptosis. A. Jurkat cells were first cultured to 1 Amol/L SR141716, 2 Amol/L SR144528,10 nmol/L PMA, or 25 Amol/L U0126 in medium containing 10% FBS in the absence or presence of 10 Amol/L THC for a total 12 hours, after which the extentof apoptosis was analyzed by TUNEL assay. TUNEL-positive cells were quantified by flow cytometric analysis. B. Jurkat cells were exposed to varyingconcentration of THC and U0126 at a fixed ratio (1:2.5) for 24 hours, after which the percentage of apoptosis was determined as described in Fig. 1A. Mediandose effect analysis was used to determine the combination index for each fraction effect. Combination indices < 1.0 correspond to synergistic interactions.Representative experiment; three independent experiments done in triplicate. C. Lysates prepared from cells pretreated with 10 nmol/L PMA or 25 Amol/LU0126 in medium containing 10% FBS in the absence or presence of 10 Amol/L THC for 12 hours were blotted and probed for phospho-ERK1/2.Representative experiment. Two additional studies yielded similar results. D. Quantitative changes in ERK1/2 phosphorylation were determined bydensitometric analysis of immunoblots. Columns, mean of triplicate determinations in five separate experiments; bars, SD. *, P < 0.05, **, P < 0.005. E.Samples collected from cells cultured as described in C for 24 hours in medium containing 10% FBS were monitored for protein levels of procaspase-10,procaspase-2, procaspase-8, Bid, procaspase-9, procaspase-3, PARP, and cleaved fragments. Representative of a minimum of three separate experiments.

Mechanism of THC-Induced Apoptosis

Mol Cancer Res 2006;4(8). August 2006

553

shown). However, no involvement of the PKA event was noted

when cells were exposed to THC in combination with H-89

(Fig. 6A). Western blot analysis (Fig. 6B) revealed that

combined treatment with THC and LY294002 (12 hours)

resulted in a down-regulation in Akt phosphorylation involving

both Thr308- and Ser473-specific phosphorylation sites, which

was more than that seen when either of these compounds was

used alone. No changes were observed in the levels of total Akt

with any treatments.

To examine whether the Akt pathway suppressed by THC

may play a role in dephosphorylation of Bad with the cell death,

Jurkat cells were stimulated with either LY294002 or U0126 at

designated concentrations. Subsequently, the cells were treated

with 10 Amol/L THC for a total 12 hours or left untreated. Thephosphorylation status of Bad was monitored by the immuno-

precipitation following by Western analyses. As shown in

Fig. 6C, THC and U0126 alone diminished the levels of

phosphorylation of Bad on site Ser112, whereas the levels of

phospho-Bad on site Ser136 remained unchanged. When cells

were exposed to THC in combination with U0126, there was a

significant reduction and essentially the complete disappear-

ance of phospho-Bad (Ser112) levels. In contrast, coadministra-

tion of THC plus LY294002 did not enhance the degree of the

dephosphorylation of Bad on site Ser136 when this was

compared with LY294002 treatment alone. Furthermore,

treatment of Jurkat cells with THC alone at the concentration

(5-15 Amol/L) of THC and the time course analyses(30 minutes to 30 hours) did not alter the level of phospho-

Bad on site Ser136 under investigation (only one data point

shown). No significant changes were observed in the levels of

phospho-Bad on site Ser155 with any treatments (Fig. 6C).

These results were confirmed using densitometric analysis

(Fig. 6D), which distinguishes the changes of Bad phosphor-

ylation after designated treatment.

Lastly, the effects of combined exposure to THC were

examined in relation to the localization of Bad. Generally, Bad

FIGURE 4. Enforced activation of MEK/ERK blocks THC-mediated apoptosis. A. Jurkat cells inducibly expressing a constitutively active, hemagglutinin-tagged MEK1 vector under the control of a tetracycline-responsive promoter were exposed to either 1 Amol/L SR141716 or 2 Amol/L SR144528 followed by10 Amol/L THC for 12 hours in the presence or absence of 2 Ag/mL doxycycline (DOX ). Percentage of apoptotic cells was then determined as described inFig. 1A. Columns, mean of three separate experiments; bars, SD. *, P < 0.05, significantly less than values obtained for the treated cells in the absence ofdoxycycline. B. Cells were treated as described in A. Proteins (prepared from cells in the absence and presence of doxycycline) were transferred onto asingle piece of nitrocellulose following fractionation using the same gel, and Western analysis was used to monitor expression of the hemagglutinin(HA ) – tagged MEK, phospho-MEK, phospho-ERK, and phospho-RSK (Thr359/Ser363) at exactly same incubation time. Levels of caspases and PARP wereimmunoassayed by culturing Jurkat cells for 24 hours as described in A. Arrows, THC-induced breakdown products or active caspases. h-Actin served as aloading control. Representative experiment. An additional study yielded equivalent results. C. Cells were treated for 24 hours as described in A, after whichDNA was isolated and subjected to agarose gel electrophoresis. Gels were stained with ethidium bromide and viewed under UV light.

Jia et al.

Mol Cancer Res 2006;4(8). August 2006

554

resides in the cytosol but translocates to the mitochondria

following death signaling. We therefore used confocal micros-

copy to study the translocation of Bad following exposure of

Jurkat cells to THC alone or in combinations with other

treatments. Double-immunofluorescence analysis with anti-Bad

antibody and mitochondria-specific dye (MitoTracker Deep

Red 633) showed a strong association of Bad with mitochon-

dria in THC-treated cells compared with vehicle-treated cells

(Fig. 6E). To further establish a link between the MEK/ERK

pathway and the translocation of Bad to the mitochondria in

THC-treated Jurkat cells, we used U0126, a potent inhibitor of

this pathway. In addition, we used LY294002 to see whether

combined treatment with LY294002 and THC could enhance

the translocation of Bad to the mitochondria. The data shown in

Fig. 6E indicated that exposure to U0126 significantly

enhanced THC-induced translocation of Bad to the mitochon-

dria and that a combination of THC plus U0126 caused marked

increase in Bad translocation to mitochondria. In addition,

treatment of Jurkat cells with LY294002 alone induced minimal

Bad translocation and THC plus LY294002 did not augment

this process when compared with THC alone (Fig. 6E). These

findings together suggested that THC induced the translocation

of Bad to mitochondria without significant association of Bad

with PI3K/Akt pathway.

To further determine whether THC induced translocation of

Bad via the interruption of Raf-1/MEK/ERK/RSK pathway,

Jurkat cells were stably transfected with a constitutively active

MEK1 construct (Mek/30). As shown in Fig. 7A, exposure to

either THC or U0126 in the absence of doxycycline resulted in

dephosphorylation of Bad on site Ser112, and these effects were

even more pronounced when cells were treated with THC plus

U0126. Similar results were obtained with a second MEK1-

inducible clone (Jurkat Mek/6; data not shown). However,

when cells were cultured in the presence of doxycycline, THC-

induced down-regulation of Bad on this site was blocked. No

changes were detected in levels of phosphorylation of Bad on

other sites when cells were cultured in the absence or presence

of doxycycline. Confocal analysis revealed that cells cultured in

the absence of doxycycline displayed a strong association of

Bad with mitochondria in THC plus U0126–treated cells,

which was more than that seen when either of these compounds

was used alone (Fig. 7B). However, when cells were cultured in

the presence of doxycycline, enforced activation of MEK/ERK/

RSK significantly blocked THC-mediated translocation of Bad

to the mitochondria, analogous to results obtained with a

second MEK1-inducible clone (Jurkat Mek/6; data not shown).

Together, these findings suggest that suppression of Raf-1/

MEK/ERK/RSK signaling pathway by THC plays an important

functional role in the induction of Bad translocation to the

mitochondria.

To confirm the requirement of Bad in THC-induced

apoptosis of Jurkat cells, we used a RNA interference approach.

It has been shown that small interfering RNA (siRNA)

consisting of 21-bp dsRNA can mediate RNA interference

effect in mammalian cells. We used two types of Bad siRNA

designated Bad siRNA-I and Bad siRNA-II as described in

Materials and Methods. Both were able to significantly reduce

Bad protein expression, whereas control siRNA had no

significant effect (Fig. 8A and C). Depletion of Bad in Jurkat

cells was found to significantly inhibit THC-mediated apoptosis

when compared with Jurkat cells transfected with control

siRNA (Fig. 8B and D). These data therefore corroborated our

earlier results showing the crucial role of Bad in THC-mediated

downstream signaling in induction of apoptosis of Jurkat cells.

DiscussionRecently, cannabinoids were shown to inhibit the prolifer-

ation of several human cancer cell lines, including leukemia

(5, 10), breast (19), prostate (20), and gliomas (16). Studies

from our laboratory showed that activation of cannabinoid

receptors on normal and transformed T cells triggers apoptosis

(10, 21). The data presented in the current study provide new

insights into the functional roles of cannabinoid receptors and

MAPK cascades in cannabinoid-mediated mitochondrial injury,

caspase activation, and cell death. MAPK pathways consist of

three parallel serine/threonine kinase (ERK, JNK, and the p38

MAPK) modules involved in the regulation of diverse cellular

events, including proliferation, differentiation, apoptosis, etc.

(22). In the current study, we investigated the effect of THC

treatment on all three modules of MAPK signaling pathways

in Jurkat cells and noted that THC-mediated cell death was

associated with interruption of the Raf-1/MEK/ERK signaling

FIGURE 5. THC-induced apoptosis is blocked by PTX. A. Log-phaseJurkat cells were pretreated with either 50 or 100 ng/mL PTX for 16 hoursin the presence or absence of 10 Amol/L THC for an additional 12 hours.The extent of apoptosis was determined as described in Fig. 1A. Columns,mean of three separate experiments done in triplicate; bars, SD. B. Cellswere cultured with 50 ng/mL PTX for 16 hours in the presence or absenceof 10 Amol/L THC for an additional 12 hours followed by immunoblotting ofwhole-cell lysates with antibodies that recognize phospho-Raf-1, Raf-1,phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2, phospho-RSK(Thr359/Ser363), RSK, phospho-p38, p38, phospho-JNK, and JNK. Levelsof caspases and PARP were immunoassayed by culturing Jurkat cells with50 ng/mL PTX for 16 hours in the presence or absence of 10 Amol/L THCfor an additional 24 hours in medium containing 10% FBS. h-Actin servedas a loading control. Representative of three independent experiments.

Mechanism of THC-Induced Apoptosis

Mol Cancer Res 2006;4(8). August 2006

555

pathway without significantly altering the p38 and JNK

modules. Consequently, we further investigated the effect of

THC on the upstream and downstream events that modulate the

ERK module of MAPK. The pronounced down-regulation of

phospho-Raf-1 was also associated with a marked reduction

in phosphorylation of MEK, a major Raf-1 substrate (23),

phospho-ERK, the primary MEK target (24), and RSK, the first

substrate of ERK (25). This process occurred early, was depen-

dent on cannabinoid receptor ligation, and proceeded upstream

of caspase activation. THC also decreased the phosphorylation

of Akt. However, no significant association of Bad trans-

location with PI3K/Akt and PKA signaling pathways was noted.

The regulation of mitochondrial membrane function and the

release of apoptotic regulatory factors from mitochondria are

key components of the apoptotic repertoire, tightly controlled

by the Bcl-2 family proteins (26, 27). There is accumulated

evidence suggesting that the MAPK pathway, through Bad

phosphorylation, plays a significant functional role in cell

survival (26). The family of RSK was among the first

cytoplasmic MAPK substrates identified (28). RSK phosphor-

ylate a variety of substrates and regulate a diverse array of

cellular functions, such as gene transcription, protein synthesis,

and cell cycle regulation (29). Thus, MAPK activates RSK,

which in turn catalyzes the phosphorylation of Bad (26). Cell

survival is associated with the phosphorylation of Bad, which,

by interacting with the scaffold protein 14-3-3 in the cytosol,

prevents its translocation to the mitochondria (30). In the

absence of survival signals, Bad is dephosphorylated, trans-

locates to the mitochondria, and interacts with Bcl-2 and

Bcl-XL, thereby preventing the survival function of Bcl-2 and

Bcl-XL (30, 31). Data from the present study showed that THC

inactivated the cytoprotective serine/threonine kinase pathway

FIGURE 6. Effects of THC on mitochondrial localization of Bad in THC-stimulated cells. A. Jurkat cells were treated with either 15 Amol/L LY294002 (LY)or 2 Amol/L H-89 in medium containing 10% FBS in the absence or presence of 10 Amol/L THC for a total 12 hours, after which the percentage of apoptoticcells were determined as described in Fig. 1A. Columns, mean of three separate experiments done in triplicate; bars, SD. *, P < 0.05, significantly less thanvalues obtained for the treated cells with THC + LY294002. B. Cells were cultured with either 15 Amol/L LY294002 or 25 Amol/L U0126 in medium containing10% FBS in the absence or presence of 10 Amol/L THC for a total 12 hours, after which Western analysis was used to monitor expression of phospho-Akt(Thr308), phospho-Akt (Ser473), and Akt. Akt served as a loading control. C. Cells were cultured as described in B, after which precleared cell lysates wereincubated overnight with mouse monoclonal anti-Bad IgG conjugated to protein A-agarose beads. Immunoprecipitates were subsequently subjected toWestern analysis to monitor the phosphorylation status of phospho-Bad (Ser112), phospho-Bad (Ser136), and phospho-Bad (Ser155). All sites were analyzedon a single blot. Cos cells were used as a positive control. Representative experiment. Two additional studies yielded equivalent results. D. Quantitativechanges in Bad phosphorylation were determined by densitometric analysis of immunoblots. Columns, mean of three independent experiments done intriplicate; bars, SD. E. Jurkat cells treated as described in B, after which cells were adhered to slides by cytospin and subjected to double staining withanti-Bad antibodies and Cy2-labeled secondary antibodies (green ) followed by a mitochondrion-specific dye (MitoTracker Deep Red 633) and then analyzedby confocal microscopy. Similar results were obtained in three independent experiments.

Jia et al.

Mol Cancer Res 2006;4(8). August 2006

556

(Raf-1/MEK/ERK/RSK) that plays an important functional role

in mediating Bad translocation. This may have caused the

localization of Bad to the mitochondria as seen following THC

treatment.

Based on these results, the effect of THC treatment on the

PI3K/AKT and PKA signaling pathways was also examined

in the regulation of Bad and cell death (32-34). Our findings

indicated that THC caused the marked reduction in the level

of phospho-Bad on site Ser112 without significantly altering

the PKA module. The effect on Bad Ser112 was more

pronounced when cells were exposed to THC in combination

with U0126 consistent with recent studies that the MAPK-

activated RSK phosphorylates Bad on Ser112 (30, 35).

Although there is down-regulation of phospho-AKT on both

sites Thr308 and Ser473 following exposure of Jurkat cells to

THC, no change was detected in phospho-Bad on the site

Ser136, which was reported to be implicated in this pathway

(32, 33). This finding was confirmed by THC dose response

and time course analysis when treated cells were examined in

phosphorylation status of Bad on this site. Moreover, confocal

analysis indicated that LY294002, an inhibitor of PI3K, failed

to enhance the degree of Bad translocation following exposure

of Jurkat cells to THC. In view of evidence linking the

dysregulation of the PI3K/Akt progression to apoptosis, it is

tempting to invoke this mechanism to explain the ability of

THC and LY294002 to induce cell death in Jurkat cells.

However, identification of the specific events responsible for

PI3K inhibitor-mediated lethality remains an elusive goal. Akt

is implicated in post-translational modification of Bad (32),

regulation of the expression of antiapoptotic proteins,

including Bcl-2 and XIAPS (36), and modulation of diverse

pathways governing cell survival decisions, including those

associated with GSK (37), mammalian target of rapamycin

(38), nuclear factor-nB (39), etc. (40-42). In this regard, thefinding that LY294002, an inhibitor of PI3K, failed to

enhance THC-mediated translocation of Bad to mitochondria

argues against a critical role for this pathway in regulation of

Bad phosphorylation.

It has long been known that cannabinoids, including THC,

can stimulate the MAPK cascade in Chinese hamster ovary

cells (13), rat and mouse hippocampus (14), rat primary

astrocytes (43), human astrocytoma cells (44), transformed

neural cells (16, 45) human breast cancer cells (19), and the

striatum (46). Furthermore, a recent report has shown evidence

indicating that THC-induced apoptosis in certain types of

leukemic cells is mediated by down-regulation of ERK (5). Our

findings represented THC-mediated ERK inactivation and cell

death in a variety of human tumor cell lines, which are

consistent with this report in the literature (5). However, the

precise role of cannabinoid receptor as a modulator of the ERK

cascade is still a matter of debate. It is generally accepted that

the activation of the ERK cascade leads to cell proliferation

(47). However, recent investigations have begun to define

situations in which ERK mediates cell cycle arrest (48),

antiproliferation (49), and apoptotic (50) or nonapoptotic (51)

death in many cell lines. In most but not all systems studied,

FIGURE 7. Enforced ex-pression of constitutively activeMEK blocks THC translocationof Bad to the mitochondria. A.Jurkat cells expressing consti-tutively active MEK (Mek/30)were exposed to U0126 (25Amol/L) and THC (10 Amol/L)alone or in combination for 12hours, after which proteins (pre-pared from cells in the absenceand presence of doxycycline)were loaded onto the sameSDS-PAGE followed by trans-ferring onto a single piece ofnitrocellulose, and the immuno-precipitates were subjected toWestern analysis to monitor thephosphorylation status of phos-pho-Bad (Ser112), phospho-Bad(Ser136), and phospho-Bad(Ser155) at same incubationtime. Representative experi-ment. Two additional studiesyielded equivalent results. B.Cell lysates were prepared fromclones (Mek/30) of Jurkat cellsas described in A, after whichcells were adhered to slides bycytospin and subjected to dou-ble staining with anti-Bad anti-bod i es and Cy2 - l abe l edsecondary antibodies (green )followed by a mitochondrion-specific dye (MitoTracker DeepRed 633) and then analyzed byconfocal microscopy. Repre-sentative of three independentexperiments.

Mechanism of THC-Induced Apoptosis

Mol Cancer Res 2006;4(8). August 2006

557

inactivation of MEK and ERK is associated with the promotion

of cell death (52, 53). The mechanism by which this occurs is

not known with certainty but may vary based on the cell type.

Except this, it may be related to perturbations in downstream

ERK targets, including the Elk (54), CREB family of

transcription factors (35), etc. Alternatively, inactivation of

ERK may prevent phosphorylation of Bad and in so doing

preserve its proapoptotic capacity through interaction with

Bcl-2/Bcl-XL (26, 27). To further define the functional role of

the MEK/ERK/RSK pathway in the translocation of Bad to

mitochondria, Jurkat cells that were stably transfected with a

constitutively active MEK construct were employed. Consistent

with this model, enforced expression of constitutively active

MEK overcame the suppression of ERK/RSK activation and

the reduction in the levels of phospho-Bad on Ser112 and

substantially protected cells from THC/U0126 lethality. In fact,

the protection effects of enforced ERK activation plays an

important role in blocking the localization of Bad to

mitochondria. However, given the pleiotropic nature of THC

action, the possibility that other downstream targets of this

agent contribute to lethality cannot be excluded. The relation

between activation of the ERK cascade and cell proliferation/

antiproliferation depends on various stimuli (55) and varies

between diverse types of cells (56, 57).

In a recent study, it was shown that C6 glioma cells exposed

to a synthetic cannabinoid, WIN 55,212-2, exhibited down-

regulation of the Akt and ERK signaling pathways before

induction of apoptosis (58). Exposure to WIN 55,212-2 caused

a decrease in phospho-Bad. In addition, Powles et al. (5)

showed that THC-induced cell death in leukemic cells was

FIGURE 8. Bad plays a crit-ical role in THC-induced apopto-sis. A and B. Jurkat cells weretransfected with Bad siRNA-I. Cand D. Cells were transfectedwith siRNA-II as described inMaterials and Methods. A andC. Bad expression level wasanalyzed by Western blottingusing antibody against Bad. Band D. Jurkat cells transfectedwith Bad siRNA were culturedwith vehicle or THC and ana-lyzed for apoptosis using TUNELassay. C. Right, signal intensitywhen compared with h-actin.Columns, mean of three inde-pendent experiments; bars, SE.D. Right, percent apoptosis.Columns, mean of three inde-pendent experiments; bars, SE.*, P < 0.05, compared withcontrols.

Jia et al.

Mol Cancer Res 2006;4(8). August 2006

558

preceded by significant changes in the expression of genes

involved in MAPK signal transduction pathways. The current

study further extends these observation in a leukemia model by

identifying Raf-1/MEK/ERK/RSK-mediated localization of

Bad to mitochondria as a critical mechanism regulating

cannabinoid-induced lethality. This was supported by the

observation that enforced expression of constitutively active

MEK/ERK resulted in the inhibition of mitochondrial Bad

localization, caspase activation, and apoptosis. Moreover,

down-regulation of Bad expression by siRNA led to marked

resistance of Jurkat cells to THC-mediated apoptosis. Although

the ERK pathway has been shown to play a pivotal role in

regulating cell growth and differentiation to growth factors,

cytokines, and phorbol esters (59), it is also weakly activated

by stress (60, 61). In contrast, JNK and p38 are weakly

activated by growth factors but are highly activated in

response to stress signals, including tumor necrosis factor,

ionizing and UV irradiation, and hyperosmotic stress, which

lead to induction of apoptosis (53, 62). For example, there is

mixed evidence for the role of ERK in influencing cell

survival of cisplatin-treated cells. Some studies have suggested

that ERK activation is associated with enhanced survival of

cisplatin-treated cells (63, 64), whereas others have shown that

elevated expression of Ras, an upstream component of the

ERK signaling pathway, leads to increased sensitivity to the

drug (65).

THC and other cannabinoids can induce apoptosis in a

variety of tumor cell lines, thereby raising the possibility of the

use of cannabinoids as novel anticancer agents (11). However,

the use of cannabinoids that activate CB1 receptors is severely

limited by their psychoactive effects. The fact that malignant

cells of the immune system express CB2 receptors that can be

targeted to induce apoptosis offers a novel approach to use CB2

select agonists as anticancer drugs with no psychoactive

properties. Because CB2 receptors are almost exclusively

expressed on immune cells, the use of CB2 select agonists

should not exert generalized toxicity that is common to other

modes of treatment, such as radiation or chemotherapy. One

possible drawback could be that use of select CB2 agonists to

kill tumor cells may also cause immunosuppression. Thus,

further studies are necessary to address the relative sensitivity

of normal and transformed immune cells to CB2 agonists

in vivo . Identifying the molecular pathways that trigger

apoptosis following ligation of cannabinoid receptors is critical

in understanding how endogenous and exogenous cannabinoids

may regulate the growth of normal and transformed immune

cells. The current study provides useful and novel information

on developing a new class of anticancer drugs by targeting

cannabinoid CB2 receptors.

Materials and MethodsCells

Jurkat, Molt4, SupT1, Hut78, and U251 glioma cell lines

were purchased from American Type Culture Collection

(Rockville, MD). The cells were cultured in RPMI 1640

supplemented with 10% fetal bovine serum (FBS; Atlanta,

Norcross, GA). They were maintained in a 37jC, 5% CO2,fully humidified incubator.

ReagentsTHC was obtained from the National Institute of Drug

Abuse (Rockville, MD). SR141716 and SR144528 were

obtained from Sanofi Recherche (Montpellier, France). PTX

and LY294002 were purchased from Sigma (St. Louis, MO).

3,3-Dihexyloxacarbocyanine iodide, U0126, PMA, and the

PKA inhibitor H-89 were purchased from Calbiochem (San

Diego, CA). Annexin V/PI was purchased from BD PharMin-

gen (San Diego, CA). The pan-caspase inhibitor Z-VAD-FMK

was purchased from R&D Systems (Minneapolis, MN) and all

agents were initially dissolved in DMSO (Sigma) as stock

solution and stored at �20jC.

Detection of THC-Induced Apoptosis In vitroJurkat cells (4 � 105 per well) were cultured in 24-well

plates in the presence or absence of designated concentrations

of THC for 12 to 24 hours. After cytospin, the cell preparations

were stained with Wright-Giemsa and viewed by light

microscopy to evaluate the extent of apoptosis as described

previously (52). The percentage of apoptotic cells was

determined by evaluating z500 cells per treatment in triplicate.To confirm the results of morphologic analysis, TUNEL

method was used as described elsewhere (66).

Flow Cytometry to Detect Apoptosis by TUNELFollowing each treatment, the cells were harvested, washed

twice with PBS, and fixed with 4% paraformaldehyde for 30

minutes at room temperature. The cells were then permeabi-

lized on ice for 2 minutes, incubated with FITC-dUTP and

terminal deoxynucleotidyl transferase (Boehringer Mannheim,

Indianapolis, IN) for 1 hour at 37jC and 5% CO2, andanalyzed using a Beckman Coulter Cytomics FC 500

(Fullerton, CA).

Determination of MMPMMP was monitored using 3,3-dihexyloxacarbocyanine

iodide as described (9). For each condition, 4 � 105 cells wereincubated in 1 mL 3,3-dihexyloxacarbocyanine iodide (40

nmol/L) at 37jC in for 15 minutes and subsequently analyzedusing a Becton Dickinson FACScan cytofluorometer (Mans-

field, MA) with excitation and emission settings of 488 and

525 nm, respectively. Control experiments documenting the

loss of MMP were done by exposing cells to 5 Amol/Lcarbamoyl cyanide m -chlorophenylhydrazone (Sigma; 15

minutes, 37jC), an uncoupling agent that abolishes the MMP.

Annexin V/PIAnnexin V/PI staining was done as described previously

(10). The cells were analyzed using a Becton Dickinson

FACScan flow cytometer.

RNA Isolation and Reverse Transcription-PCRRNA was isolated from f1 � 107 cells using the RNeasy

Mini kit (Qiagen, Valencia, CA) according to the recommen-

dations from the manufacturer. DNA was removed by the

RNase-free DNase set (Qiagen) following RNA isolation. RNA

concentration was determined spectrophotometrically, and the

integrity of the each preparation was verified by agarose gel

Mechanism of THC-Induced Apoptosis

Mol Cancer Res 2006;4(8). August 2006

559

electrophoresis. cDNAwas synthesized by reverse transcription

of 50 ng total RNA as template for first-strand synthesis using

the Sensiscript RT kit (Qiagen). All PCR was prepared using

MasterAmp PCR Premix F (Epicenter Technologies, Madison,

WI) according to the recommendations from the manufacturer

and using Platinum Taq DNA polymerase (Invitrogen, Carlsbad,

CA). Human CB1 was amplified using primers HCB1U (5¶-CG-TGGGCAGCCTGTTCCTCA-3¶) and HCB1L (5¶-CATGC-GGGCTTGGTCTGG-3¶), which yield a product of 403 bp.Human CB2 was amplified using primers HCB2U (5¶-CGCC-GGAAGCCCTCATACC-3¶) and HCB2L (5¶-CCTCATTCG-GGCCATTCCTG-3¶), which yield a product of 522 bp. h-Actinwas used as a positive control, with primers BAU (5¶-AAGG-CCAACCGTGAAAAGATGACC-3¶) and BAL (5¶-ACCGCTC-GTTGCCAATAGTGATGA-3¶), with a product size of 427 bp.PCR was carried out using the following variables: 95jC for10 seconds, 59jC for 20 seconds, and 72jC for 45 seconds for35 cycles followed by a final 1 minute at 72jC in an AppliedBiosystems GeneAmp 9700 (Foster City, CA). The resulting

PCR products were separated on a 1.2% agarose gel.

Immunoblot AnalysisImmunoblotting was done as described previously (52). The

source of antibodies was as follows: caspase-2 (Alexis, San

Diego, CA); Bid, phospho-ERK1/2 (Thr202/Tyr204), JNK,

phospho-JNK, phospho-38, phospho-p38 MAPK, phospho-

Akt (Thr308), phospho-Akt (Ser473), Akt, and Bad (C-7; Santa

Cruz Biotechnology, Santa Cruz, CA); Bad, phospho-Bad

(Ser112), phospho-Bad (Ser136), phospho-Bad (Ser155), caspase-

3, caspase-8, caspase-9, caspase-10, ERK1/2, PARP, MEK1/2,

phospho-MEK1/2, p90RSK, phospho-p90RSK (Thr359/Ser363),

phospho-p90RSK (Ser380), and phospho-p90RSK (Thr573; Cell

Signaling Technology, Danvers, MA); c-Raf-1, phospho-c-

Raf-1 (Biosource, Camarillo, CA); and h-actin (Sigma). Celllysates were prepared and the concentration of the protein was

measured by using standard Bradford assays. The proteins were

fractionated in SDS-PAGE and transferred onto polyvinylidene

difluoride membranes using a dry-blot apparatus (Bio-Rad,

Hercules, CA). The membrane was incubated in blocking

buffer for 1 hour at room temperature followed by incubation in

primary antibody at 4jC overnight. The membrane was thenwashed thrice (10-15 minutes) with washing buffer (PBS-0.2%

Tween 20) and incubated for 1 hour in horseradish peroxidase–

conjugated secondary antibody (Cell Signaling Technology,

Inc.) in blocking buffer. The membranes were then washed

several times and incubated in developing solution (equal

volume of solutions A and B; Enhanced Chemiluminescence

Western Blotting Detection Reagents, Amersham Biosciences,

Little Chalfont, United Kingdom) and signal was detected using

ChemiDoc System (Bio-Rad). Densitometric analyses of the

Western blots were done with UN-SCAN-IT (Silk Scientific,

Orem, UT) software digitizer technology.

ImmunoprecipitationAfter drug treatments, cells were washed with PBS and

incubated for 10 minutes in lysis buffer [150 mmol/L NaCl,

50 mmol/LTris-HCl (pH 8.0), 1 mmol/L EDTA, 1% (v/v) NP40,

0.25% (w/v) sodium deoxycholate, 1 mmol/L NaF, 1 mmol/L

sodium pyrophosphate, 100 Amol/L Na3VO4, 1 mmol/L

phenylmethylsulfonyl fluoride, 10 Ag/mL aprotinin, 10 Ag/mLleupeptin]. Precleared by incubation with protein A-agarose

bead slurry, cell lysates (2 Ag/AL total cell protein) were incu-bated overnight with mouse monoclonal anti-Bad IgG or rabbit

polyclonal anti-Bad IgG conjugated to agarose beads. Immuno-

precipitates were captured by addition of protein A-agarose. The

agarose beads were collected by centrifugation, washed twice

in ice-cold lysis buffer, boiled in Laemmli sample buffer, and

subjected to SDS-PAGE and subsequent immunoblot analysis.

Tet-On-Inducible Jurkat Cell LinesStably transfected Jurkat clones that inducibly expressed

constitutively active MEK1 were generated as described (52).

To test for the induced expression of the hemagglutinin-tagged

MEK1, stable clones were left untreated or were treated for

designated periods with 2 Ag/mL doxycycline (Sigma),harvested, and analyzed for expression of the appropriate

protein by Western blot as described above.

Transfection of Jurkat Cells with Mouse Bad siRNAJurkat cells (5 � 106) were transfected with 1.7 Amol/L

human siRNA (5¶-GAAGGGACUUCCUCGCCCGTT-3¶; 5¶-CGGGCGAGGAAGUCCCUUCTT-3¶; designated siRNA-I;Cell Signaling Technology, Inc., Danvers, MA) using nucleo-

fection of Jurkat cells with Transfection Reagent kit and

Nucleofactor II electroporation system following the protocols

of the company (Amaxa, Inc., Gaithersburg, MD). Jurkat cells

were also transfected with human-specific control siRNA

(1.7 Amol/L) conjugated with fluorescein (Cell SignalingTechnology, Inc., Danvers, MA) and pmaxGFP plasmid

(2 Ag; Amaxa). In a separate experiment, 5 � 106 Jurkat cellswere also transfected with human Bad siRNA (siGENOME

SMARTpool reagent M-003870-02; 3 Ag; Dharmacon RNATechnologies, Lafayette, CO; designated siRNA-II) using

nucleofection of Jurkat cells with Transfection Reagent kit

and Nucleofactor II electroporation system following the

protocols of the company. As a control, 5 � 106 Jurkat cellswere transfected with human-specific negative control SiGLO

RISK-free siRNA (3 Ag; designated control siRNA-II)unconjugated or conjugated with Cy-3 (Pool D-001206-13-

05; Dharmacon RNA Technologies) and pmaxGFP plasmid

(2 Ag). Transfected Jurkat cells were cultured for 48 hours incomplete medium at 37jC and 5% CO2. We observed >90%cell viability after transfection. To examine the efficiency of

transfection, we observed pmaxGFP plasmid-transfected Jurkat

cells under fluorescent microscope and observed >50% cells

expressing green fluorescent protein. We also examined the

expression of green fluorescent protein by performing flow

cytometry. Jurkat cells, untransfected or transfected with control

siRNA or human Bad siRNA, were treated with vehicle or

THC, and 24 hours after treatment, cells were harvested and

apoptosis was determined by performing TUNEL assay and

using flow cytometry.

Immune Complex Kinase Assay (a NonradioactiveMethod)

ERK activity was measured in THC-treated cell lysates using

p44/42 MAPK Assay kit (Cell Signaling Technology, Inc.,

Danvers, MA).

Jia et al.

Mol Cancer Res 2006;4(8). August 2006

560

DNA Fragmentation AssayFor qualitative assessment of internucleosomal DNA

fragmentation, DNA was extracted from cell lysates after the

appropriate treatment and subjected to agarose gel electropho-

resis as described previously (67).

Confocal MicroscopyConfocal microscope images were captured on a LSM 510

microscope with a �60, 1.3 NA Apochromat objective (CarlZeiss, Inc., Thornwood, NY). Jurkat cells were grown as

described above on a 24-well plate (Corning, Corning, NY).

After treatment, confocal microscopy of cells was done after

double staining with primary antibody against Bad and

mitochondrion-specific dye (MitoTracker Deep Red 633;

Molecular Probes, Inc. Eugene, OR) as indicated in individual

experiments. The excitation wavelength for Bad and Mito-

Tracker Deep Red 633 were 490 and 644 nm, respectively.

Statistical AnalysisThe significance of differences between experimental

conditions was determined using the two-tailed Student’s t

test. To characterize synergistic or antagonistic interactions

between agents, median dose effect analysis (68) was employed

using a commercially available software program (Calcusyn,

Biosoft, Ferguson, MO).

References1. Watson SJ, Benson JA, Jr., Joy JE. Marijuana and medicine: assessing thescience base: a summary of the 1999 Institute of Medicine report. Arch GenPsychiatry 2000;57:547–52.

2. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of acannabinoid receptor and functional expression of the cloned cDNA. Nature1990;346:561–4.

3. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of aperipheral receptor for cannabinoids. Nature 1993;365:61–5.

4. Pertwee RG. Pharmacology of cannabinoid CB1 and CB2 receptors.Pharmacol Ther 1997;74:129– 80.

5. Powles T, te Poele R, Shamash J, et al. Cannabis-induced cytotoxicity inleukemic cell lines: the role of the cannabinoid receptors and the MAPK pathway.Blood 2005;105:1214–21.

6. Vincent BJ, McQuiston DJ, Einhorn LH, Nagy CM, Brames MJ. Review ofcannabinoids and their antiemetic effectiveness. Drugs 1983;25 Suppl 1:52 –62.

7. Roth MD, Baldwin GC, Tashkin DP. Effects of D-9-tetrahydrocannabinol onhuman immune function and host defense. Chem Phys Lipids 2002;121:229– 39.

8. Do Y, McKallip RJ, Nagarkatti M, Nagarkatti PS. Activation throughcannabinoid receptors 1 and 2 on dendritic cells triggers NF-nB-dependentapoptosis: novel role for endogenous and exogenous cannabinoids in immuno-regulation. J Immunol 2004;173:2373–82.

9. Lombard C, Nagarkatti M, Nagarkatti SP. Targeting cannabinoid receptors totreat leukemia: role of cross-talk between extrinsic and intrinsic pathways in D(9)-tetrahydrocannabinol (THC)-induced apoptosis of Jurkat cells. Leuk Res 2005;29:915– 22.

10. McKallip RJ, Lombard C, Fisher M, et al. Targeting CB2 cannabinoidreceptors as a novel therapy to treat malignant lymphoblastic disease. Blood 2002;100:627 –34.

11. Guzman M. Cannabinoids: potential anticancer agents. Nat Rev Cancer 2003;3:745– 55.

12. Bouaboula M, Poinot-Chazel C, Bourrie B, et al. Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1.Biochem J 1995;312:637 –41.

13. Rueda D, Galve-Roperh I, Haro A, Guzman M. The CB(1) cannabinoidreceptor is coupled to the activation of c-Jun N-terminal kinase. Mol Pharmacol2000;58:814–20.

14. Derkinderen P, Ledent C, Parmentier M, Girault JA. Cannabinoids activate

p38 mitogen-activated protein kinases through CB1 receptors in hippocampus.J Neurochem 2001;77:957– 60.

15. Rueda D, Navarro B, Martinez-Serrano A, Guzman M, Galve-Roperh I. Theendocannabinoid anandamide inhibits neuronal progenitor cell differentiationthrough attenuation of the Rap1/B-Raf/ERK pathway. J Biol Chem 2002;277:46645 –50.

16. Galve-Roperh I, Sanchez C, Cortes ML, del Pulgar TG, Izquierdo M,Guzman M. Anti-tumoral action of cannabinoids: involvement of sustainedceramide accumulation and extracellular signal-regulated kinase activation. NatMed 2000;6:313 –9.

17. Gomez Del Pulgar T, De Ceballos ML, Guzman M, Velasco G. Cannabinoidsprotect astrocytes from ceramide-induced apoptosis through the phosphatidyli-nositol 3-kinase/protein kinase B pathway. J Biol Chem 2002;277:36527 –33.

18. Eisenmann KM, VanBrocklin MW, Staffend NA, Kitchen SM, Koo HM.Mitogen-activated protein kinase pathway-dependent tumor-specific survivalsignaling in melanoma cells through inactivation of the proapoptotic protein bad.Cancer Res 2003;63:8330– 7.

19. Melck D, Rueda D, Galve-Roperh I, De Petrocellis L, Guzman M, Di MarzoV. Involvement of the cAMP/protein kinase A pathway and of mitogen-activatedprotein kinase in the anti-proliferative effects of anandamide in human breastcancer cells. FEBS Lett 1999;463:235 –40.

20. Ruiz L, Miguel A, Diaz-Laviada I. D9-Tetrahydrocannabinol inducesapoptosis in human prostate PC-3 cells via a receptor-independent mechanism.FEBS Lett 1999;458:400– 4.

21. McKallip RJ, Lombard C, Martin BR, Nagarkatti M, Nagarkatti PS. D(9)-Tetrahydrocannabinol-induced apoptosis in the thymus and spleen as amechanism of immunosuppression in vitro and in vivo . J Pharmacol Exp Ther2002;302:451 –65.

22. Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature2001;410:37–40.

23. Hipskind RA, Baccarini M, Nordheim A. Transient activation of RAF-1,MEK, ERK2 coincides kinetically with ternary complex factor phosphorylationand immediate-early gene promoter activity in vivo . Mol Cell Biol 1994;14:6219– 31.

24. Troppmair J, Bruder JT, Munoz H, et al. Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and12-O -tetradecanoylphorbol-13-acetate requires Raf and is necessary for transfor-mation. J Biol Chem 1994;269:7030– 5.

25. Smith JA, Poteet-Smith CE, Malarkey K, Sturgill TW. Identification of anextracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, asequence critical for activation by ERK in vivo . J Biol Chem 1999;274:2893– 8.

26. Korsmeyer SJ. BCL-2 gene family and the regulation of programmed celldeath. Cancer Res 1999;59:1693–700s.

27. Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med 2000;6:513 –9.

28. Frodin M, Gammeltoft S. Role and regulation of 90 kDa ribosomal S6 kinase(RSK) in signal transduction. Mol Cell Endocrinol 1999;151:65 –77.

29. Gavin AC, Ni Ainle A, Chierici E, Jones M, Nebreda AR. A p90(rsk) mutantconstitutively interacting with MAP kinase uncouples MAP kinase fromp34(cdc2)/cyclin B activation in Xenopus oocytes. Mol Biol Cell 1999;10:2971– 86.

30. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation ofdeath agonist BAD in response to survival factor results in binding to 14-3-3 notBCL-X(L). Cell 1996;87:619–28.

31. Scheid MP, Duronio V. Dissociation of cytokine-induced phosphorylation ofBad and activation of PKB/Akt: involvement of MEK upstream of Badphosphorylation. Proc Natl Acad Sci U S A 1998;95:7439 –44.

32. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couplessurvival signals to the cell-intrinsic death machinery. Cell 1997;91:231– 41.

33. del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 1997;278:687–9.

34. Zhou XM, Liu Y, Payne G, Lutz RJ, Chittenden T. Growth factors inactivatethe cell death promoter BAD by phosphorylation of its BH3 domain on Ser155.J Biol Chem 2000;275:25046– 51.

35. Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME. Cellsurvival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 1999;286:1358–62.

36. Leverrier Y, Thomas J, Mathieu AL, Low W, Blanquier B, Marvel J. Role ofPI3-kinase in Bcl-X induction and apoptosis inhibition mediated by IL-3 or IGF-1in Baf-3 cells. Cell Death Differ 1999;6:290– 6.

37. Pap M, Cooper GM. Role of glycogen synthase kinase-3 in the

Mechanism of THC-Induced Apoptosis

Mol Cancer Res 2006;4(8). August 2006

561

phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem 1998;273:19929 –32.

38. Edinger AL, Thompson CB. Akt maintains cell size and survival byincreasing mTOR-dependent nutrient uptake. Mol Biol Cell 2002;13:2276 –88.

39. Gelfanov VM, Burgess GS, Litz-Jackson S, et al. Transformation ofinterleukin-3-dependent cells without participation of Stat5/bcl-xL: cooperationof Akt with Raf/Erk leads to p65 nuclear factor nB-mediated antiapoptosisinvolving c-IAP2. Blood 2001;98:2508–17.

40. Vanhaesebroeck B, Waterfield MD. Signaling by distinct classes ofphosphoinositide 3-kinases. Exp Cell Res 1999;253:239– 54.

41. Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection: more than just aroad to PKB. Biochem J 2000;346:561 –76.

42. Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathwayin human malignancy. Cell Signal 2002;14:381–95.

43. Sanchez C, Galve-Roperh I, Rueda D, Guzman M. Involvement ofsphingomyelin hydrolysis and the mitogen-activated protein kinase cascade inthe D9-tetrahydrocannabinol-induced stimulation of glucose metabolism inprimary astrocytes. Mol Pharmacol 1998;54:834–43.

44. Bouaboula M, Bourrie B, Rinaldi-Carmona M, Shire D, Le Fur G, Casellas P.Stimulation of cannabinoid receptor CB1 induces krox-24 expression in humanastrocytoma cells. J Biol Chem 1995;270:13973 –80.

45. Sanchez C, Galve-Roperh I, Canova C, Brachet P, Guzman M. D9-Tetrahydrocannabinol induces apoptosis in C6 glioma cells. FEBS Lett 1998;436:6 –10.

46. Valjent E, Pages C, Rogard M, Besson MJ, Maldonado R, Caboche J. D9-Tetrahydrocannabinol-induced MAPK/ERK and Elk-1 activation in vivo dependson dopaminergic transmission. Eur J Neurosci 2001;14:342–52.

47. Derkinderen P, Enslen H, Girault JA. The ERK/MAP-kinases cascade in thenervous system. Neuroreport 1999;10:R24 –34.

48. Pumiglia KM, Decker SJ. Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc Natl Acad Sci U S A 1997;94:448–52.

49. York RD, Yao H, Dillon T, et al. Rap1 mediates sustained MAP kinaseactivation induced by nerve growth factor. Nature 1998;392:622 –6.

50. Mohr S, McCormick TS, Lapetina EG. Macrophages resistant toendogenously generated nitric oxide-mediated apoptosis are hypersensitive toexogenously added nitric oxide donors: dichotomous apoptotic responseindependent of caspase 3 and reversal by the mitogen-activated protein kinasekinase (MEK) inhibitor PD 098059. Proc Natl Acad Sci U S A 1998;95:5045–50.

51. Murray B, Alessandrini A, Cole AJ, Yee AG, Furshpan EJ. Inhibitionof the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity. Proc Natl Acad Sci U S A 1998;95:11975–80.

52. Jia W, Yu C, Rahmani M, et al. Synergistic antileukemic interactions between17-AAG and UCN-01 involve interruption of RAF/MEK- and AKT-relatedpathways. Blood 2003;102:1824–32.

53. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effectsof ERK and JNK-p38 MAP kinases on apoptosis. Science 1995;270:1326 –31.

54. Gille H, Kortenjann M, Thomae O, et al. ERK phosphorylation potentiatesElk-1-mediated ternary complex formation and transactivation. EMBO J 1995;14:951 –62.

55. Marshall CJ. Signal transduction. Taking the Rap. Nature 1998;392:553– 4.

56. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathwaysmediated by ERK, JNK, p38 protein kinases. Science 2002;298:1911–2.

57. Howe AK, Aplin AE, Juliano RL. Anchorage-dependent ERK signaling—mechanisms and consequences. Curr Opin Genet Dev 2002;12:30 –5.

58. Ellert-Miklaszewska A, Kaminska B, Konarska L. Cannabinoids down-regulate PI3K/Akt and Erk signalling pathways and activate proapoptotic functionof Bad protein. Cell Signal 2005;17:25 –37.

59. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. CurrOpin Cell Biol 1997;9:180 –6.

60. Guyton KZ, Liu Y, Gorospe M, Xu Q, Holbrook NJ. Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury.J Biol Chem 1996;271:4138–42.

61. Aikawa R, Komuro I, Yamazaki T, et al. Oxidative stress activatesextracellular signal-regulated kinases through Src and Ras in cultured cardiacmyocytes of neonatal rats. J Clin Invest 1997;100:1813–21.

62. Chen YR, Wang X, Templeton D, Davis RJ, Tan TH. The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and g radiationduration of JNK activation may determine cell death and proliferation. J BiolChem 1996;271:31929– 36.

63. Hayakawa J, Ohmichi M, Kurachi H, et al. Inhibition of extracellular signal-regulated protein kinase or c-Jun N-terminal protein kinase cascade, differentiallyactivated by cisplatin, sensitizes human ovarian cancer cell line. J Biol Chem1999;274:31648–54.

64. Persons DL, Yazlovitskaya EM, Cui W, Pelling JC. Cisplatin-inducedactivation of mitogen-activated protein kinases in ovarian carcinoma cells:inhibition of extracellular signal-regulated kinase activity increases sensitivity tocisplatin. Clin Cancer Res 1999;5:1007–14.

65. Fokstuen T, Rabo YB, Zhou JN, et al. The Ras farnesylation inhibitor BZA-5B increases the resistance to cisplatin in a human melanoma cell line. AnticancerRes 1997;17:2347 –52.

66. Chen D, McKallip RJ, Zeytun A, et al. CD44-deficient mice exhibitenhanced hepatitis after concanavalin A injection: evidence for involvement ofCD44 in activation-induced cell death. J Immunol 2001;166:5889– 97.

67. Jarvis WD, Povirk LF, Turner AJ, et al. Effects of bryostatin 1 and otherpharmacological activators of protein kinase C on 1-[h-D-arabinofuranosyl]cyto-sine-induced apoptosis in HL-60 human promyelocytic leukemia cells. BiochemPharmacol 1994;47:839–52.

68. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: thecombined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul1984;22:27– 55.

Jia et al.

Mol Cancer Res 2006;4(8). August 2006

562